Since the first observations of cells as the “fundamental unit” of all living organisms, ranging from unicellular microbes such as Escherichia coli or Saccharomyces cerevisiae to multicellular, highly differentiated plants and animal, scientists have invested considerable effort in developing methods for isolating and investigating cells. Some populations of broadly similar cells can be obtained in large numbers by controlled fermentation in the case of microbes, or by tissue isolation in fluids such as spermatozoa or classes of blood cells in mammals, or laterally by cell culture technologies.
Magnetic beads are a popular format for capturing and purifying cells. In this technology, a suspension of beads is mixed with a suspension of cells. The magnetic beads can contain a tag or chemical entity selective for cells or for a particular cell type within the sample. After the cells become associated with the magnetic beads, a magnet is used to collect the magnetic beads with the associated cells. The magnetic beads may be resuspended several times with wash solutions to remove unbound cells. Finally, a solution can be used to release the cells from the beads and a magnet separates the magnetic beads from the cells.
However, magnetic beads have a number of drawbacks. First, they can negatively impact cell viability. Cell damage or death can occur when magnetic beads (with cells attached) are captured by a magnet. During this magnetic capture process, cells can be damaged or killed by crushing or squeezing as the beads are pressed together. This magnetic capture process must be performed several times endangering the cells. Second, magnetic bead methods have lengthy processing times and physically batter the cells which can result in cell clumping and death. Other common problems encountered with magnetic bead methods include low cell yield and impure cell populations.
There exists a need for a column technology that rapidly captures high concentrations of cells, particularly viable cells and then recovers the cells at high purity for research, detection and for other uses.
In the instant invention, it was discovered that cells can be captured on a column. In some embodiments, the captured cells remain viable and viable cells can be eluted from the column. Remarkably, cell capture can be performed by flowing the cells back and forth through a column. Previously, it was shown that small analytes such as proteins could be captured on a column using back and forth flow however, it was quite surprising that cells could withstand this treatment and remain viable.
The columns and methods described herein can be used to purify a desired cell type from a heterogeneous biological sample. Alternatively, contaminants can be captured and the desired cell type can flow through the column.
Captured cells can be manipulated on the column using a number of different strategies. In a second invention described herein, living cells captured on a column solid phase can be used as a stationary phase for liquid chromatography. After cell capture, the cells attached to the column can serve as a stationary phase and reagents, analytes or biomolecules can be passed through the column. In some embodiments, these reagents or biomolecules can be passed through the column using back and forth flow. The interaction of these entities with the stationary phase can be examined. If it is desired, the stationary phase comprised of cells, the cell-based stationary phase may be recovered and analyzed.